1. Field of the Invention
This invention relates to the field of medical genetics. More specifically, the invention provides for therapeutic agents for Alzheimer""s Disease and methods of screening for therapeutic agents for Alzheimer""s disease that are based on affecting alpha-2-macroglobulin function and expression.
2. Related Art
Alzheimer""s disease (AD) is a devastating neurodegenerative disorder that affects more than 4 million people per year in the US (Dxc3x6beli, H., Nat. Biotech. 15:223-24 (1997)). It is the major form of dementia occurring in mid to late life: approximately 10% of individuals over 65 years of age, and approximately 40% of individuals over 80 years of age, are symptomatic of AD (Price, D. L., and Sisodia, S. S., Ann. Rev. Neurosci. 21:479-505 (1998)).
The first recognized clinical symptom of AD is usually the loss of short-term memory (Schellenberg, G. D., Proc. Nati. Acad Sci. USA 92:8552-559 (1995)). Other common symptoms include abnormal judgement and behavior, and difficulty with language, orientation, problem solving, calculations, and visuospacial perception (Price, D. L., and Sisodia, S. S., Ann. Rev. Neurosci. 21:479-505 (1998); Schellenberg, G. D., Proc. Natl. Acad. Sci. USA 92:8552-559 (1995)). These symptoms often worsen until cognitive function is almost entirely lost, and the patient cannot function independently (Schellenberg, G. D., Proc. Natl. Acad. Sci. USA 92:8552-559 (1995); Price, D. L., and Sisodia, S. S., Ann. Rev. Neurosci. 21:479-505 (1998)). By late stages of the disease patients typically lack verbal ability, cannot recognize people, and are incontinent and bed-ridden (Price, D. L., and Sisodia, S. S., Ann. Rev. Neurosci. 21:479-505 (1998); Sloane, P. D., Am. Family Phys. 58: 1577-86 (1998)).
Known risk factors for AD include age, genetic predisposition, abnormal protein (xcex2-amyloid) deposition in the brain, and certain environmental factors such as head injury, hypothyroidism, and a history of depression. The majority of AD patients do not exhibit symptoms until their seventies (Price, D. L., and Sisodia, S. S., Ann. Rev. Neurosci. 21:479-505 (1998)). However, individuals who have inherited particular genetic defects often exhibit symptoms in midlife (Price, D. L., and Sisodia, S. S., Ann. Rev. Neurosci. 21:479-505 (1998)). This latter type of AD, called early-onset familial AD (FAD), accounts for 5-10% of AD cases, and has been linked to defects in three different genes, APP, PSEN1, PSEN2 (Blacker, D. and Tanzi, R. E., Archives of Neurology 55:294-296 (1998)). Mutations in these genes lead to increased production of the amyloidogenic xcex2-amyloid peptide (Axcex2) (Citron, M., et al, Nature Medicine 3:67-72 (1997); Suzuki, N., et al., Science 264:1336-1340 (1994)).
The most prevalent form of AD, called late-onset AD (LOAD), accounts for approximately 90% of AD cases, and has been genetically linked to APOE and LRP (Kang, D. E., et al., Neurology 49:56-61 (1997); Kounnas, M. Z., el al., Cell 82:331-340 (1995)). Recently, another gene, the alpha-2-macroglobulin gene (A2M), was found to be linked to LOAD (Blacker, D., et al., Nature Genetics 19:357-360 (1998)). Carriers of a particular mutation in A2M were discovered to be at increased risk of AD. This mutation is a pentanucleotide deletion at the 5xe2x80x2 splice site of the second exon encoding the bait region of alpha-2-macroglobulin (xcex12M), and is referred to as the A2M-2 genotype. The A2M-2 genotype is present in 30% of the population (Blacker, D., et al., Nature Genetics 19:357-360 (1998)). The A2M-2 pentanucleotide deletion is a predisposing factor for AD.
Presently, there is no cure for AD on the horizon and its incidence is increasing as the population ages (Price, D. L., and Sisodia, S. S., Ann. Rev. Neurosci. 21:479-505 (1998)). Due to the lateness in life of the onset of AD symptoms, the ability to delay onset by as little as 5 years could decrease the number of AD patients by as much as 50% (Marx, J., Science 273:50-53 (1996)). With the large number of people already affected, and projected to be affected by AD, a drug that could merely delay the onset of AD would be very valuable.
Therapeutic agents based on predisposing factors of AD might be able to prevent, delay or slow progression of the disease. However, presently, available treatments are primarily aimed at treatment of the symptoms of the disease (Enz, A., xe2x80x9cClasses of drugs,xe2x80x9d in: Pharmacotherapy of Alzheimer""s Disease, Gauthier, S., ed., Martin Dunitz, publ., Malden, Mass. (1998)). These AD drugs offer only modest success, and at most, merely slow the progression of the disease (Delagarza, V. W., Am. Family Phys. 58:1175-1182 (1998); Enz, A., xe2x80x9cClasses of drugs,xe2x80x9d in: Pharmacotherapy of Alzheimer""s Disease, Gauthier, S., ed., Martin Dunitz, publ., Malden, Mass. (1998)). Presently approved and investigational drugs for treating AD can be characterized as those whose actions enhance neurotransmitter effect, or those believed to protect neurons (Delagarza, V., Am. Family Phys. 58:1175-1182 (1998)). The most well known drugs in the first category are the cholinesterase inhibitors, such as tacrine (Cognex(trademark)) and doneprezil (Aricept(trademark)), both of which have been approved by the FDA (Delagarza, V., Am. Family Phys. 58:1175-1182 (1998); Sloan, P., Am. Family Phys. 58:1577-1586 (1998)). Tacrine and doneprezil are only modestly effective (Sloan, P., Am. Family Phys. 58:1577-1586 (1998)), and are associated with unpleasant side effects including nausea and vomiting (Delagarza, V., Am. Family Phys. 58:1175-1182 (1998)). Several neuro-protective drugs are under investigation for the treatment of AD, including estrogen, vitamin E, selegiline and non-steroidal anti-inflammatory drugs (NSAIDs) (Sloan, P., Am. Family Phys. 58:1577-1586 (1998); Delagarza, V., Am. Family Phys. 58:1175-1182 (1998)). None of these drugs have been approved yet for the treatment of AD, and each has significant drawbacks, including negative side-effects, or association with increased risk of other diseases. (Sloan, P., Am. Family Phys. 58:1577-1586 (1998); Delagarza, V., Am. Family Phys. 58:1175-1182 (1998); Enz, A., xe2x80x9cClasses of drugs,xe2x80x9d in: Pharmacotherapy of Alzheimer""s Disease, Gauthier, S., ed., Martin Dunitz, publ., Malden, Mass. (1998)).
Thus, there is a need for new AD therapeutic agents, especially those based on predisposing factors of AD. In addition, there is a need for drug screening systems to aid in developing these therapeutic agents.
Based on the finding, described herein, that the A2M-2 deletion leads to the production of altered xcex12M RNA transcripts and proteins, strategies aimed at replacing or supplementing normal xcex12M function and activities, and/or at suppressing defective xcex12M function in the brain may serve as a means for therapeutically preventing, treating, or even reversing AD neuropathogenesis. In addition, these strategies may be useful for treating other pathologies associated with defective xcex12M function. Moreover, methods described herein may be used to screen for these therapeutic agents. Thus, the invention provides for new therapeutic agents for AD, for pharmaceutical compositions containing these therapeutic agents, for methods of using these therapeutic agents, and for methods of screening for these therapeutic agents.
The first aspect of the invention is to provide for a therapeutic agent for Alzheimer""s Disease, where the agent can replace or supplement xcex12M function, or can suppress the expression of A2M-2. A molecule that can bind to Axcex2 and to LRP may be able to promote clearance of Axcex2 through LRP mediated endocytosis. Thus, one embodiment of the invention is an anti-LRP-Axcex2 molecule having an Axcex2 binding domain, and an LRP binding domain. In a preferred embodiment of the invention, this molecule is a peptide.
In one embodiment of the invention the peptide is an anti-LRP-Axcex2 peptide having an Axcex2 binding domain composed of 10-50 contiguous residues of SEQ ID NO:6, and an LRP binding domain comprising 10-50 contiguous residues of SEQ ID NO:8, which encompass residues 1366-1392 of SEQ ID NO:8. In another embodiment of the invention, the anti-LRP-Axcex2 peptide has an Axcex2 binding domain with an amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, and SEQ ID NO:26; and an LRP binding domain composed of the amino acid sequence of SEQ ID NO:10. In yet another embodiment of the invention, the anti-LRP-Axcex2 peptide has an Axcex2 binding domain with an amino acid sequence selected from the group consisting of SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, and SEQ ID NO:26; and an LRP binding domain composed of 10-50 contiguous residues of SEQ ID NO:8.
The Axcex2 binding domain may be connected to the LRP binding domain of the anti-LRP-Axcex2 molecule by a covalent bond, linker molecule, or linkerless polyethylene glycol. In a preferred embodiment, the Axcex2 and LRP binding domains are connected by a peptide bond. In another preferred embodiment of the invention, the Axcex2 and LRP binding domains are connected by a peptide composed of 1-20 glycine residues.
In another embodiment, the anti-LRP-Axcex2 peptide has the amino acid sequence of SEQ ID NO:14. Alternatively, the anti-LRP-Axcex2 peptide has an Axcex2 binding domain having an amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, and SEQ ID NO:26; an LRP binding domain having the amino acid sequence of SEQ ID NO:10; and a linker connecting the Axcex2 binding domain to the LRP binding domain.
In addition, the invention provides for pharmaceutically acceptable salts of the anti-LRP-Axcex2 peptide and for nucleic acid molecules encoding the anti-LRP-Axcex2 peptide.
Another embodiment of the invention relates to a nucleic acid molecule encoding an anti-LRP-xcex2 peptide, where the Axcex2 binding domain is encoded by 30-150 contiguous nucleotides of SEQ ID NO:5, and the LRP binding domain is encoded by 30-150 contiguous nucleotides of SEQ ID NO:7. In another embodiment of the invention, the region of the nucleic acid molecule encoding the Axcex2 binding domain has a nucleotide sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, and SEQ ID NO:25; and the region encoding the LRP binding domain has the nucleotide sequence of SEQ ID NO:9. In yet another embodiment of the invention, the region of the nucleic acid molecule encoding the Axcex2 binding domain has a nucleotide sequence selected from the group consisting of SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, and SEQ ID NO:25; and the region encoding the LRP binding domain is encoded by 30-150 contiguous nucleotides of SEQ ID NO:7. In another embodiment of the invention, the nucleic acid molecule has the nucleotide sequence of SEQ ID NO:13.
The region encoding the Axcex2 binding domain may be connected to the region encoding the LRP binding domain of the nucleic acid molecule by a phosphodiester bond. Alternatively, these regions may be connected by a nucleotide encoding a linker peptide. In a preferred embodiment of the invention, the connecting nucleotide encodes 1-20 glycine residues.
In addition, the invention relates to nucleic acid molecules having at least 95% homology to these nucleic acid molecules.
Another embodiment of the invention relates to a nucleic acid molecule that is a first polynucleotide that hybridizes to a second polynucleotide that is complementary to the nucleic acid molecules described above. In another embodiment of the invention, the nucleic acid molecule is a first polynucleotide that hybridizes to a second polynucleotide that is complementary to the nucleotide sequence of SEQ ID NO:13. In yet another embodiment of the invention, the hybridizing conditions for the hybridization of the first and second polynucleotides are as follows: (a) incubate overnight at 42xc2x0 C. in a solution consisting of 50% formamide, 5xc3x97SSC, 50 mM sodium phosphate (pH 7.6), 5xc3x97Denhardt""s solution, 10% dextran sulfate, and a 20 xcexcg/ml denatured, sheared salmon sperm DNA; and (b) wash at 65xc2x0 C. in a solution consisting of 0.1xc3x97SSC.
A related embodiment of the invention is a pharmaceutical composition containing an anti-LRP-Axcex2 molecule, and one or more pharmaceutically acceptable carriers. In addition, the invention provides for a pharmaceutical composition containing an anti-LRP-Axcex2 peptide, or a pharmaceutically acceptable salt thereof. In a preferred embodiment, the pharmaceutical composition contains an anti-LRP-Axcex2 peptide having an amino acid sequence selected from the group consisting of SEQ ID NO:4 or SEQ ID NO:14, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers. The invention also relates to a method of combating Alzheimer""s Disease in a subject by administering an anti-LRP-Axcex2 molecule, or a pharmaceutically acceptable salt thereof. In a preferred embodiment, the anti-LRP-Axcex2 molecule is a peptide. In another preferred embodiment, the anti-LRP-Axcex2 peptide is a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:4 or SEQ ID NO:14, or a pharmaceutically acceptable salt thereof.
The invention also relates to an A2M-2 antisense oligonucleotide designed to target A2M-2 RNA. In one preferred embodiment of the invention, the A2M-2 antisense oligonucleotide is designed to target A2M-2 heteronuclear RNA. In another preferred embodiment, the A2M-2 antisense oligonucleotide is designed to target A2M-2 mRNA. In one embodiment of the invention, the A2M-2 antisense oligonucleotide designed to target A2M hnRNA has the nucleotide sequence of SEQ ID NO:27. The A2M-2 antisense oligonucleotide is preferably from 8-50 nucleotides in length, and more preferably is 15-30 nucleotides in length, and is most preferably 15 nucleotides in length. Thus, in another preferred embodiment of the invention an A2M-2 antisense oligonucleotide designed to target A2M-2 hnRNA has the nucleotide sequence of the last 15-30 contiguous nucleotides of SEQ ID NO:27. In another embodiment of the invention the A2M-2 antisense oligonucleotide designed to target A2M-2 has the sequence of nucleotides 36-50 of SEQ ID NO:27 or of nucleotides 20-50 of SEQ ID NO:27. The invention also relates to a pharmaceutical composition containing an A2M-2 antisense oligonucleotide, and one or more pharmaceutically acceptable carriers. In addition, the invention relates to a method of combating Alzheimer""s Disease in a subject by administering the A2M-2 antisense oligonucleotide.
The invention also provides for a viral vector carrying a transgene encoding xcex12M, or an anti-LRP-Axcex2 peptide. in a preferred embodiment of the invention, the viral vector carries a gene encoding xcex12M. In another preferred embodiment of the invention, the gene encoding xcex12M has the nucleotide sequence of nucleotides 44-4465 of SEQ ID NO:1. The invention also relates to a viral vector carrying a gene encoding an anti-LRP-Axcex2 peptide. In another preferred embodiment of the invention, the viral vector is an adeno-associated virus. In addition, the invention provides for a pharmaceutical composition containing the viral vector, and one or more pharmaceutically acceptable carriers, and for a method of combating Alzheimer""s Disease in a subject by administering the viral vector.
The second aspect of the invention is to provide for a method of screening for therapeutic agents for Alzheimer""s Disease that can replace or supplement xcex12M function, or can suppress the expression of A2M-2. One embodiment of the invention is a method of screening for a therapeutic agent for AD by incubating a cell that is heterozygous or homozygous for the A2M-2 allele in the presence of a test agent, and then determining whether the ratio of normal to aberrant A2M mRNA has increased relative to the ratio of normal to aberrant A2M mRNA found in cells untreated with the test agent. In one preferred embodiment of this method, the cells are glioma cells. In another preferred embodiment, the cells are hepatoma cells. In yet another preferred embodiment of the invention, the cells are heterozygous for the A2M-2 allele.
In a related embodiment of this method, S1 nuclease is used to determine the ratio of normal to aberrant A2M mRNA, and the probe used is complementary to a nucleotide encoding A2M (SEQ ID NO:1). Thus, in one embodiment of the invention, S1 nuclease analysis using a probe complementary to SEQ ID NO:1, where the probe encompasses nucleotides 2057-2284 of SEQ ID NO:1, is used to determine whether the ratio of normal to aberrant A2M mRNA has increased. In a preferred method of the invention, the probe used in the S1 nuclease analysis is 300 bp long. In another embodiment of the invention, the probe used in the S1 nuclease analysis is complementary to nucleotides 2024-2323 of SEQ ID NO:1.
Alternatively, RT PCR analysis is used to determine whether the ratio of normal to aberrant A2M mRNA has increased. In a preferred method of RT PCR analysis, the primers are designed to amplify a region of A2M encompassing exons 17-18. In a more preferred method of RT PCR analysis, the amplified region of A2M encompassing exons 17-18 is 300 bp long. In another embodiment of the invention, the primers used for the RT PCR analysis are designed to amplify nucleotides 2052-2289 of SEQ ID NO:1. Another embodiment of the invention relates to the use of a first primer having a nucleotide sequence complementary to nucleotides 2024-2038 of SEQ ID NO:1, and a second primer having the nucleotide sequence of nucleotides 2309-2323 of SEQ ID NO:1 for the RT PCR analysis.
The invention also provides for a method of screening for a therapeutic agent for Alzheimer""s disease by incubating xcex12M with a test agent, and then determining whether the treated xcex12M has undergone a conformational change, or determining whether the treated xcex12M can bind to LRP. In a preferred embodiment of the invention, the xcex12M treated with a test agent is tetrameric xcex12M. In another preferred embodiment of the invention, an xcex12M electrophoretic mobility assay is ued to determine whether the treated xcex12M has undergone a conformational change. In another embodiment of the invention, an ELISA is used to determine whether the treated xcex12M can bind to LRP. In a related embodiment of the invention, the ELISA includes the following steps in sequential order: incubating LRP in a well coated with anti-LRP IgG, incubating the well with treated xcex12M, incubating the well with anti-xcex12M IgG conjugated to an enzyme, and incubating the well with a substrate for the enzyme. In an alternative embodiment, the ELISA includes the following steps in sequential order: incubating a well coated with LRP with treated xcex12M, incubating the well with anti-xcex12M IgG conjugated to an enzyme, and incubating the well with the substrate for the enzyme. In another embodiment, the ELISA includes the following steps in sequential order: incubating treated xcex12M in a well coated with an anti-xcex12M IgG specific for activated xcex12M, incubating the well with an anti-xcex12M IgG conjugated to an enzyme, and incubating the well with a substrate for the enzyme. In another embodiment of the invention, immunoblotting with anti-LRP IgG and anti-xcex12M IgG is used to determine whether the treated xcex12M can bind to LRP. In yet another embodiment of the invention, a test for the ability of the treated xcex12M to undergo LRP mediated endocytosis is used to determine whether the treated xcex12M can bind to LRP. In another embodiment of the invention, a test for the ability of the treated xcex12M to undergo LRP mediated degradation is used to determine whether the treated xcex12M can bind to LRP.